Structural features in messenger RNA that affect the fidelity and efficiency of translational initiation will be probed by introducing defined mutations into a cloned preproinsulin gene carried on a eukaryotic expression vector. The pBR322-based shuttle vector has the rat preproinsulin II coding sequence linked to the SV40 early promoter. Synthesis of proinsulin will be monitored during transient expression of the mutant plasmids in monkey (COS) cells. The optimal sequence context for initiation by eukaryotic ribosomes will be determined by introducing mutations in positions -1 to -5; i.e. directly preceding the AUG initiator codon. Once the optimal -1 to -5 sequence has been defined, we will determine whether that sequence still facilitates initiation when it is moved slightly farther upstream from the initiator codon. There is preliminary evidence that the natural ribosome binding site of the rat preproinsulin II gene is a very efficient initiation site. To determine whether sequences extending even beyond the -5 position contribute to the efficiency of that site, the upstream region will be mutagenized. In other experiments, we will determine whether translation is affected by introducing a long hairpin structure upstream from the AUG initiator codon. In another construct, a hairpin structure will be created that directly involves the AUG codon. The efficiency of translating pro-insulin from such a construct will help to distinguish between a model in which the 40S ribosomal subunit binds upstream and moves down to the AUG codon (presumably unfolding the mRNA as it advances) versus a model in which involvement of an AUG codon in a base-paired stem makes it completely inaccessible to ribosomes. To follow up the recent discovery that eukaryotic ribosomes can "reinitiate" following a terminator codon, we will determine whether the efficiency of reinitiation is affected by the distance between the terminator codon and the next AUG triplet. We will try to find conditions that allow reinitiation to occur in vitro. These experiments should enhance our understanding of how eukaryotic ribosomes select the appropriate site in mRNA for initiation of protein synthesis. We may also gain insight into some of the features that modulate translational efficiency in eukaryotes.